**5. Xenobiotics**

In several areas where agricultural activities occur, the possibility of finding pesticide-free areas has decreased. Because of this, an increase in the density of apiaries has been observed with the systematic appearance of bee diseases. The continuous exposure of bees to xenobiotics (agricultural pesticides and veterinary products) is responsible for the presence of these compounds in recycled waxes [26]. Although the information available is just related to reports from a limited group of countries, there is enough evidence that the presence of these compounds in the surrounding areas of beehives, as well as in the composition of products obtained from those apiaries, may have a long-term effect on the reproductive health of beekeepers, even farmer workers, or consumers [27].

#### **6. Residues of pesticides: analytical methods**

The use of pesticides in agriculture is allowed under strict regulations, but nowadays there is enough evidence about the negative effects of those products over bee health. In that way, the presence of pesticides in the honey content may be detected owing to direct contamination from beekeeping practices or by indirect contamination from environmental sources [28]. Since the mid-1990s, several beekeepers from different parts of United States and Europe reported high colony losses caused by a phenomenon known as Colony Collapse Disorder (CCD) [29]. One of the key factors of this disorder has been the use of neonicotinoids, a class of neurotoxic pesticides. Some studies have showed its effects by killing bees after expositions of these compounds, and there **is** evidence of damages at sublethal doses of neonicotinoids over their nervous systems affecting foraging abilities, navigation, learning communication, and memory. Also, the suppression of the immune systems of bees may be caused by neonicotinoids [30, 31]. In 2013, the European Food Safety Authority (EFSA) identified several risks posed to bees by three neonicotinoid insecticides: Clothianidin, Imidacloprid, and Thiamethoxam as seed treatment or as granules, with particular regard to their acute and chronic effects on bee colony survival and development. For this reason, the use of those products has been restricted by European Union. In addition, these restrictions are extensive for treated seeds with those pesticides (EFSA Journal 2013; 3066; 3067; 3068) [32]. It has been described that neonicotinoids are not the only group of insecticides with negative effects. The organophosphate and organochlorine also cause damage to the bees [33].

Among the methods used for the detection and identification of both insecticides and pesticides, the most useful technique is HPLC with mass spectrometry [34]. As previously indicated for pesticides, the critical step in those determinations is related to the pretreatments of samples before chromatographic analysis. One alternative methodology was developed for 13 pesticides detected in 40 samples of honeys from Poland. In this case, all the samples were subjected to liquid–liquid extraction process on a diatomaceous earth support. The main difficulty in this methodology was the matrix effects over the percentages of recoveries of pesticides (63–117%), when the samples were fortified for assessing the success of this extraction process [35]. At the present time, the analyses include the QuEChERS method followed by dispersive solid-phase extraction (d-SPE). This is simple sample preparation technique

recommended for pesticides detection in a wide variety of food and agricultural products. Several studies have achieved satisfactory results in the determination of a long list of pesticides belonging to different classes such as organophosphates, triazoles, carbamates, dicarboximides, dinitroanilines, and neonicotinoids in honeybee bodies, honey, and bee pollen. The advantage of QuEChERS is the improvement of precision of measurements and percentages of recovery of pesticides after analysis without a matrix effect of samples affecting the reliability of results. This permits better sensitivity and lower detection limits for each pesticide [36–38].

The group of compounds corresponding to agronomic pesticides and veterinary products is wide and growing day by day. Despite this, there is a consensus on the analytical methods available for their detection and quantification. **Table 1** presents a list of susceptible compounds that can be identified in bee products with their main methodologies for their extraction, detection, and/or quantification (adapted from [39]).








#### **Table 1.**

*List of pesticides analyzed in honey and beeswax samples. DL: Detection Limit; QL: Quantification Limit.*
